**6. NPs for overcoming immunosuppression**

**4.4. NPs in combination for delivering cancer vaccines**

56 Immunization - Vaccine Adjuvant Delivery System and Strategies

elevated mouse survival, compared to control PBS-treated animals.

(SLLs) of 50 nm, both of which were loaded with a model Ag and NH<sup>4</sup>

**5. NPs for delivering DNA and mRNA vaccines**

The VADs based on various NPs that are engineered with different adjuvants such as PRRas and tumor Ags in combination for delivery of vaccines to the same immune cells have a great potential in provoking immune responses, generating increased duration and speed of immune response, regulating Ag-antibody response, and amplifying immunogenicity of weak Ags [12]. For example, it is reported that the application of poly(γ-glutamic acid)-based NPs for the delivery of model Ag (OVA) and toll-like receptor 3 (TLR3) agonist poly (I:C) (polyinosinic-polycytidylic acid) in targeting the LNs significantly enhanced the antitumor immunity against EG7-OVA (EL-4 thymoma cells transfected with chicken albumin cDNA) in tumor-bearing mice [57]. Recently, Molino et al. designed a biomimetic approach for eliciting antitumor responses through engineering the viral-mimicking protein NP vaccine, which is pyruvate dehydrogenase E2 protein NP (of 50 nm) conjugated to gp100 epitope (melanomaassociated Ag) and CpG [58]. The CpG-gp-E2 NPs remarkably increased the proliferation of Ag-specific CD8+ T cells and production of IFN-γ and dramatically enhanced the population of CD8+ T cells in dLNs, resulting in the delayed onset of tumor growth in mice as well as

It should be pointed out that delivery of vaccine Ags and adjuvants to target tissues or cells by a VADS is also, to a great extent, dictated by NP properties, such as particle size and surface charge, which may be appropriately engineered for improving their delivery efficiency [59]. For example, the NP-based vaccines could be either delivered actively to the lymph nodes by DCs in target tissues or transported by the interstitial flow into the lymphatics, depending mainly on NP size and surface properties such as PEGylation and charge due to the upper limit of pore size of the lymphatic capillaries and cell uptake of NPs relevant to surface properties of both [12, 60]. Wang's group engineered two types of multifunctional liposomes, the mannosylated lipid A-liposomes (MLLs) with a size of 200 nm and the stealth lipid A-liposomes

into microneedles, forming the proSLL/MLL-constituted microneedle array (proSMMA) as a multifunctional VADS [12]. Mice vaccinated with proSMMAs by vaginal mucosa patching administration established robust Ag-specific humoral and cellular immunity at both systemic and mucosal levels, especially, in the reproductive and intestinal ducts, under the revealed mechanism that the MLLs reconstituted from the administered microneedles were mostly taken up by vaginal mucosa resident DCs, whereas the recovered SLLs trafficked directly to dLNs wherein they are to be picked up by macrophages, proving the size of NPs

Using DNA and mRNA for intracellular production of oncogenic proteins or peptides as tumor Ags becomes an attractive strategy for developing cancer vaccines thanks to the advances in biotechnology which allows gene encoding proteins of interest to be easily manufactured in batch and be further modified with nucleic acid sequences that encode for proteins with immunostimulatory functions, for example, flagellin and a toll-like receptor

as an important parameter in controlling the in vivo fate of the delivered vaccines.

HCO<sup>3</sup>

and fabricated

With great advances in immunology and oncology, several mechanisms, involving multiple immune components, have been identified to contribute to tumor immune escape, as summarized by Chabanon and coauthors as these including [69]: (1) reduction of MHC-I molecule expression in malignant cells, resulting in decreased antigen presentation and consequently reduced detection by CTLs; (2) induction of immune cell apoptosis by cancer cells through the expression of death signals; (3) release of a variety of immune-modulatory molecules such as IL6 and IL10 by tumor cells in the microenvironment to induce immunosuppressive Tregs while inhibiting the activity of CTLs; (4) secretion of TGF-β, COX-2 (cyclooxygenase-2), and PGE2 (prostaglandin E2) by tumor cells inhibiting DC differentiation and maturation while favoring the establishment of an immunosuppressive tumor microenvironment; (5) upregulated expression of immune checkpoint ligands to activate immune checkpoint receptors providing co-inhibitory signals to CD4+ and CD8+ T cells preventing them from building a specific antitumor immune response.

Among these elements involved in cancer resistance, immune checkpoints are regulators of the immune system to provide pathways crucial for self-tolerance and thus play an important role both in the prevention of autoimmunity refraining the immune system from attacking cells indiscriminately under normal physiological conditions and in the regulation of immune reaction to avoid tissue damages during the pathogenic infection. Under normal conditions, immune checkpoints function via the interaction between a receptor expressed on T cells and its ligand located at the surface of APCs to generate a co-stimulatory signal, which triggers either the activation or inhibition of T cells. Presently, two major checkpoints have been clearly identified to regulate T cell activation: (i) the CD28/CTLA-4 axis, which activates T cells upon engagement of CD28 with CD80 and CD86, and conversely inhibits T cells when CTLA-4 is engaged and (ii) the PD-1 axis, which provides a strong inhibitory signal following binding of PD-L1 or PD-L2 to the PD-1 receptor [70]. Contrary to CTLA-4, PD-1 is thought to act predominantly in the tumor microenvironment, where PD-L1 is overexpressed by multiple cell types, including dendritic cells, M2 macrophages, and tumor-associated fibroblasts [71]. Thus, immune checkpoints and pathways, unfortunately, are also utilized by cancer cells as a key mechanisms to realize immune escape through upregulated expression of immune checkpoint ligands and as such deregulation of immune checkpoint signaling to suppress T cell activity in tumor microenvironment, a phenomenon that has been observed in multiple malignancies. Moreover, immune checkpoint molecules have been shown to promote the epithelial-mesenchymal transition of tumor cells and the acquisition of tumor-initiating potential and resistance to apoptosis and antitumor drugs, as well as the propensity to disseminate and metastasize, and thus have been increasingly considered as a crucial target for cancer immunotherapy given their potential for use in multiple types of cancers. Notably, as opposed to other immunebased approaches developed to fight cancers, immune checkpoint blockers (ICBs) have displayed significant therapeutic successes in many solid tumors and hematologic malignancies, as exampled by several anti-PD-(L)1-based drugs, such as the anti-CTLA-4 ipilimumab (by Bristol-Myers Squibb), the anti-PD-1 pembrolizumab (by Merck), and the anti-PD-L1 atezolizumab (by Genentech/Roche), durvalumab (by AstraZeneca/MedImmune), and avelumab (by Pfizer), all of which have already been approved for cancer immunotherapy [69].

with relevant functional molecules. Yang et al. engineered folic acid-modified NPs with polyethyleneimine (PEI) derivatives and demonstrated that PD-L1 siRNA-loaded PEI NPs efficiently inhibited PD-L1 expression on SKOV-3-Luc tumor cells, resulting in sensitizing tumor cells to T cell killing in vitro [74]. Considering cancer recurrence after surgical resection remains still a significant challenge and platelets can accumulate in wound sites and interact with circulating tumor cells (CTCs) triggering inflammation and repair processes in the remaining tumor microenvironment, Gu's group engineered the anti-PD-L1 antibodyconjugated platelets (P-aPDL1) which were employed to reduce postsurgical tumor recurrence and metastasis [75]. In mouse models bearing partially removed primary melanomas (B16-F10) or 4T1 (triple-negative breast carcinomas), B16-F10 effectively released anti-PD-L1 upon platelet activation by platelet-derived microparticles and remarkably prolonged overall mouse survival after surgery by reducing the risk of cancer regrowth and metastatic spread, suggesting engineered platelets an efficient VADS which can facilitate the delivery of the immunotherapeutic anti-PD-L1 to the surgical bed and target CTCs in the bloodstream to

Vaccines Developed for Cancer Immunotherapy http://dx.doi.org/10.5772/intechopen.80889 59

Summarily, the VADSs based on various NPs that are engineered to bear therapeutic functions are promising in targeted delivery of the immunomodulatory agents to offset the immunosuppressive effects generated in tumor microenvironment and to rehabilitate the defensive immunity, maximizing the efficacy of cancer immunotherapy while minimizing side effects.

In recent years various types of NPs have been designed as a VADS for delivery of vaccines that are aimed for cancer immunotherapies and have shown great promise in curing refractory tumors which can never be obtained by conventional clinical measures, such as chemotherapeutics, surgery, and radiation. The NP-based cancer VADS possesses numerous advantages, including high safety profile and thus good compliance, high stability, diverse administration routes, and ease in modification with functional molecules as well as large-scale production, and bears also disadvantages including mainly relatively weak immunostimulatory capacity and low intracellular especially intranuclear delivery efficiency, which may be hopefully overcome by elaborate design with adjuvants such as PRRas and multifunctional molecules. Nevertheless, the NP-based cancer VADS proves able to successfully elicit antitumor immunity both in vitro and in vivo through, in particular, targeting APCs and draining lymph nodes, engendering lysosome escape, and modulating immunosuppression and represents

This work was financially supported by the National Natural Science Foundation of China (Grant numbers 81703449) and partially by the Department of Science & Technology of Anhui

Province for the Natural Science Research Project (Grant number 1708085QH195).

new directions in developing efficient tools for cancer immunotherapy.

improve the objective response rate.

**7. Conclusions**

**Acknowledgements**

However, with the current antibody-based immune checkpoint therapy, the nonspecific accumulation of antibody in the normal organs and tissues may ignite overreactive immune responses, which may even damage the body and cause severe side effects [72]; suggesting targeting delivery may provide beneficial effects even in the antibody-based immunotherapy. Recent studies have shown that a diverse set of NPs that have been engineered to improve delivery efficiency of immune checkpoint modulators which possess the potency in enhancement of the anticancer efficacy of the immune checkpoint blockade-based immunotherapy. Using a common procedure of water-in-oil-in-water emulsion, Wang's group formulated cationic NPs loaded with CTLA4 siRNA (siCTLA4) which was to modulate immune suppression mechanism [73]. The siCTLA4-NPs delivered siRNA into the T cells reducing mRNA and protein levels of CTLA4 upon the T cell activation in vitro and, when systemically given to mice, significantly increased the number of both CD4+ T cells and CD8+ T cells, whereas the number of CD4+ FOXP3+ regulatory T cells were decreased, resulting in the inhibited tumor growth and prolonged survival rate of B16 mouse melanoma model. PD-L1 is expressed on a variety of tumor cells, such as melanoma, NSCLC, ovarian cancer, head and neck cancer, B cell lymphoma, and thymic cancer and therefore is another attractive target for immune checkpoint modulation, which can be realized using tumor-targeted delivery system loaded with relevant functional molecules. Yang et al. engineered folic acid-modified NPs with polyethyleneimine (PEI) derivatives and demonstrated that PD-L1 siRNA-loaded PEI NPs efficiently inhibited PD-L1 expression on SKOV-3-Luc tumor cells, resulting in sensitizing tumor cells to T cell killing in vitro [74]. Considering cancer recurrence after surgical resection remains still a significant challenge and platelets can accumulate in wound sites and interact with circulating tumor cells (CTCs) triggering inflammation and repair processes in the remaining tumor microenvironment, Gu's group engineered the anti-PD-L1 antibodyconjugated platelets (P-aPDL1) which were employed to reduce postsurgical tumor recurrence and metastasis [75]. In mouse models bearing partially removed primary melanomas (B16-F10) or 4T1 (triple-negative breast carcinomas), B16-F10 effectively released anti-PD-L1 upon platelet activation by platelet-derived microparticles and remarkably prolonged overall mouse survival after surgery by reducing the risk of cancer regrowth and metastatic spread, suggesting engineered platelets an efficient VADS which can facilitate the delivery of the immunotherapeutic anti-PD-L1 to the surgical bed and target CTCs in the bloodstream to improve the objective response rate.

Summarily, the VADSs based on various NPs that are engineered to bear therapeutic functions are promising in targeted delivery of the immunomodulatory agents to offset the immunosuppressive effects generated in tumor microenvironment and to rehabilitate the defensive immunity, maximizing the efficacy of cancer immunotherapy while minimizing side effects.
